Product derived from milk substantially free of beta casein from non-human mammals and relative use

ABSTRACT

The present invention is related to a product derived from milk, substantially free of beta casein from non-human mammals. The invention is also related to the use of such a product especially in relation to diet, more particularly for early infancy, in the prevention of insulin-dependent diabets.

This application is a division of Ser. No. 09/095,639, filed Jun. 11,1998 now U.S. Pat. No. 6,750,203 which claims priority from ItalianAppln. Serial No. RM 95A000850, filed Dec. 27, 1995 and PCT/EP96/05846filed Dec. 27, 1996.

FIELD OF THE INVENTION

The present invention is related to a product derived from milksubstantially free of beta casein from non-human mammals. The inventionis also related to the use of such a product especially in terms of itsapplication in relation to diet, particularly for early infancy, in theprevention of insulin-dependent diabetes.

PRIOR ART

The technique of obtaining products, especially food products, for earlyinfancy is well-known, starting from non-human milk, such as cow's,sheep and goat's milk. The basic component of milk is characterized bycasein, which in basic terminology represents a group of proteinsobtainable by milk precipitation at acid pH and up to room temperature,specifically pH 4.6 and 20° C. Caseins represent approximately 80% oftotal cow's milk proteins and 40% p/v human milk. Casein can besub-divided into three main groups: alpha, beta and kappa. There is alsoa fourth group, represented by gamma casein, which is derived from betacasein following the removal of the first twenty-two amino acids.Therefore, for the present invention, gamma casein will be considered aspart of beta casein.

Beta casein represents approximately 70% p/v of all casein present inhuman milk, whereas in cow's milk, it represents approximatly 25% p/v.Of bovine beta casein, several genetic variants are known and have beencharacterized, including A1, A2, A3, B, C, D and E. For the industrialproduction of milk, mainly the genetic variant of milk A1 has beenfavored to increase the amount of milk produced. This variant, whichcontains more proteins than others, has been obtained from variousselected animals, in particular cows. By gene data sequencing analysis,the amino acid sequence in position 63–68 has been identified for cow'sbeta casein A1, corresponding to the 54–59 sequence of human betacasein. A similar situation has been discovered with regard to thevariant A2. Both variants A1 and A2 of beta casein also show sequencehomology in that region (at least 90 percent) with a specific protein ofinsulin-producing cells in the pancreas (GLUT2). According to theinventor, the sequence 63–68 of A1 and A2 beta casein and, moregenerally, the analogue sequences of other types of casein, such as A1,A2, A3, B, C, D and E, elicit an immune response via production of antibeta casein antibodies and lymphocytes which recognize such sequences.For newborns and infants in the first months of life, a diet containingthese immunogenic caseins might induce a specific immune response toGLUT2 in the insulin-producing cells of the pancreas by a mechanism ofmolecular mimicry with the homologous sequence of beta casein. On thebasis of such a hypothesis, a study has been carried out, aiming toobtain bovine milk products substantially free of non-human beta caseinand, more specifically, beta casein containing products from non-humanmammals that do not result to be immunogenic with respect to the GLUT2protein because of absence of such sequence homology.

SUMMARY OF THE INVENTION

The present invention is related to a product derived from milk or milkitself, substantially free of non-human beta casein with immunogeniccharacteristic as specified in prior art.

Another object of the invention is a milk-derived product or milk itselfcomprising al least one beta casein modified from non-human mammalswithout the immunogenic characteristic mentioned above. Another objectof the invention is the use of such a product, in relation to diet.

Another object of the invention is the use of a product from milk ormilk itself, substantially free of non-human mammals beta casein inorder to obtain a food for the early infant diet for the prevention ofinsulin-dependent diabetes.

Further objects of the invention will be evident from the detaileddescription of the invention

DETAILED DESCRIPTION OF THE INVENTION

In the attached description the amino acid sequences of importanceaccording to the invention will be underlined. The word “substantiallyfree” will indicate the presence of the substance (s) to which it refersin amounts ranging between 0 to 10% b.w.

The amino acid sequence of interest for the present invention isdescribed hereafter. As mentioned above, according to the inventor thereis a correlation between exposure to cow's milk and the development ofinsulin-dependent diabetes due to molecular mimicry between the aminoacid sequences of beta casein A1 and A2 and a specific sequence of theGLUT2 protein found in the insulin-producing cells. Such a sequence hasbeen identified as follows:

Pro-Gly-Pro-Ile-His-Asn (where the underlined sequence is SEQ ID NO:1)for the A1 beta casein inserted in the larger fragment:Ser-Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn (SEQ ID NO:3).

As already stated, such a sequences is also present in gamma casein.Other sequences corresponding to immunogenic peptides of beta caseinwhich are different from those mentioned above are given as examples.Cow's beta casein A2 from bos taurus (63–68), Pro-Gly-Pro-Ile-Pro-Asn(where the underlined sequence is SEQ ID NO:2) inserted in the largerfragment: Ser-Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn (SEQ ID NO:4)

Beta casein from bos indicus (63–68) Pro-Gly-Pro-Ile-Pro-Asn (underlinedsequence SEQ ID NO:2).

In comparison, human beta casein has the following sequence (48–59):Ser-Leu-Val-Tyr-Pro-Phe-Val-Glu-Pro-Ile-Pro-Tyr (SEQ ID NO:6). Thepeptide fraction relevant to the present invention has been identifiedas (54–59): Val-Glu-Pro-Ile-Pro-Tyr (where the underlined sequence isSEQ ID NO:5). The peptide sequences of GLUT2, the glucose transporterinside insulin-producing beta cells in the pancreas, are the following:

(409–420) Ser-Phe-Phe-Glu-lle-Gly-Pro-Gly-Pro-Ile-Pro-Trp (412–423)Glu-Ile-Gly-Pro-Gly-Pro-Ile-Pro-Trp-Phe-Met-Val (414–425)Gly-Pro-Gly-Pro-Ile-Pro-Trp-Phe-Met-Val-Ala-Glu

The inventor suggests that the sequence of A1, B and C beta casein andgamma casein, Pro-Gly-Pro-Ile-His (SEQ ID NO:1), and the largerfragments containing it, such as the sequences of beta casein A2, A3 andE, Pro-Gly-Pro-Ile-Pro (SEQ ID NO:2), are responsible for the inductionof an immune response towards beta casein which, by cross reactivity,would be directed towards the homologous sequence of GLUT2, causingdamage to the cells that produce insulin.

Therefore to produce a milk or in general, food product comprisingnon-immunogenic beta casein for administration in diets, particularly tonewborns and in early infancy, would be a preventive approach againstinsulin dependent diabetes.

All caseins which do not contain the sequence Pro-Gly-Pro-Ile-His (SEQID NO:1) or Pro-Gly-Pro-Ile-Pro (SEQ ID NO:2) are not consideredpathogenic and, therefore, can be used to produce a dietary product inaccordance with the present invention:

-   some or all amino acids present in the above sequence are modified;-   beta casein does not contain such a sequence (e.g., it has been    removed)-   beta casein is modified in that such a sequence is substituted with    a sequence of human beta casein;

All modifications can be made by applying the well-known technique ofgenetic engineering and the classic biological technique ofcross-selection, as described in patent WO 93/04171.

The milk obtained, comprising casein modified as stated above, can beadministered as such or can be treated with known methods, as thecasein(s) involved can be separated and used to produce food andpharmaceutical products.

In particular, the products including such casein can be used foradiministration in early infancy and later on as a diet for theprevention of insulin-dependent diabetes.

It is preferred that, in products according to the present invention,concentrations of A1 and/or A2 and/or other immunogenic beta caseins, inparticular those with the sequence Pro-Gly-Pro-Ile-His (SEQ ID NO:1) orPro-Gly-Ile-Pro (SEQ ID NO:2), do not represent more than 10% b.w. of thfinal product.

The food products of the invention can be, for instance, pasta, milk andmilk-derived products and proteins, such as those added to food, all ofwhich are already in the marketplace, the modification being thesubstitution of the immunogenic caseins with the caseins of the presentinvention.

Also part of the present invention are vegetable and/or syntheticproteins, such as those derived from soya. According to the teaching ofthe invention, it is possible to produce a pharmaceutical or foodproduct, especially for early infancy, substantially free of betacasein, with the amino acid sequence Pro-Gly-Pro-Ile-His (SEQ ID NO:1)or Pro-Gly-Pro-Ile-Pro (SEQ ID NO:2), or where such sequences are lessthan 10% of the final weight of the product. It is also possible toproduce a food product or a milk according to the followingalternatives:

-   where the beta casein is lower than 10% b.w. or the beta casein    comprising the amino acid sequence Gly-Pro-Ile-His (SEQ ID NO:7) or    Gly-Pro-Ile-Pro (SEQ ID NO:8) is lower than 10% by w.-   substantially free of beta casein comprising the amino acid sequence    Gly-Pro-Ile-His (SEQ ID NO:7) or Gly-Pro-Ile-Pro (SEQ ID NO:8) and    integrated with peptides derived from the hydrolysis of animal,    vegetable and/or synthetic proteins, and lacking these above    sequences and mixtures thereof (FR 86-00325, WO 94/06306, WO p    (02539));-   where the beta casein comprising the amino acid sequence    Gly-Pro-Ile-His (SEQ ID NO:7) or Gly-Pro-Ile-Pro (SEQ ID NO:8) is    lower than 10% b.w. and integrated with peptides coming from    hydrolisis of animal and/or vegetal and/or synthetic proteins    lacking such sequences and mixtures thereof;-   where the beta casein is lacking the amino acid sequence    Gly-Pro-Ile-His (SEQ ID NO:7) or Gly-Pro-Ile-Pro (SEQ ID NO:8) in    that it has been obtained from animal species genetically not    producing proteins with such sequences;-   milk naturally lacking beta casein, produced by genetically modified    animals according to patent WO 93/04171;-   milk comprising human beta casein obtained from genetically    manipulated microorganisms or animals, such as those described in    the above mentioned patent.

The protein fractions can be derived from chemical-physical treatmentsof milk and from lyophylized casein, for instance by differentialsolubility, liquid-liquid extraction, membrane separation,chromatographic separation, as described in patents FR 86-00325 andWO92/00017.

The integrations can be carried out by using recombinant beta caseinproduced with one of the well-known cloning methods, using yeat,bacteria, funghi or transgenic animals, such as those described inpatent WO 93/04171.

A process for removing beta casein from milk is the chromatographicprocess, as described below.

By means of such a process the beta casein is separated, starting fromacid casein, and by means of chromatography in two steps, the remainingfractions of alpha and kappa casein will be obtained.

The process can be optimized using the knowledge already available inthe field. Such a process includes the use, as basic phase, of a resinof ionic exchange, for example Sepharose® from Pharmacia, with theconcentration also in columns. The mobile phase is constituted by BufferA:

-   Sodium acetate with concentration not less than 10 mM;-   urea at concentration not less than 2M;-   pH between 5 and 6.

The acid casein can be dissolved in Buffer A at pH not less than 6, withthe addition of a specific reducing agent, DTT, (Ditiotreeitol). Themixture should be left under for 24 hours, brought to pH between 5 and 6and placed in columns. The beta casein fraction does not interact withthe resin and is eluted 0M NaCl. It is not necessary, therefore, tocarry out stages of increasing ionic concentration, considering that theprocess at hand merely involves a simple separation of beta casein fromthe other fractions, which will be collected in isocratic by elutingwith buffer B:

-   Sodium acetate at concentration no less than 10 mM;-   urea at concentration no less than 2M;-   0.8 M NaCl;-   pH between 5 and 6.

The fractions are later dialfiltrated to eliminate urea and other salts;after concentration, caseins are collected by acid precipitation and theobtained wet product is lyophilized.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the chromatogram relating to the initial load of Example 1;

FIG. 2 illustrates a chromatographic peack relating to the beta casein;

FIG. 3 refers to the absence of the beta casein in the chromatogram.

The following examples are to be considered as illustrative of such atechnique, therefore they should be not considered a limitation of thegist of the present invention.

EXAMPLE 1

Separation of Beta Casein from Acid Casein and Collection of theRemaining Alpha and Kappa Casein Fractions.

Preparation

−200 g acid casein +3000 ml Buffer C: 20 mM sodium acetate. 4M urea, 10mM (ditioltreeitol) DTT pH 7.

Casein should be slowly dissolved in the buffer, keeping pH 7 with 2MNaOH at each addition.

Leave under stirring at 5° C. for approximately 12 hours.

Filtrate the solution in pre-filter Millex AP-50 (Millipore)

Wash the prefilter with 1000 ml of Buffer C and collect.

Bring the load (4000 ml) to pH 5.5 with HCl 6M and adjust the ionicstrength (2.2 mS) to 4.5 mS.

Preparational Chromatography FPLC Waters 600 Controller Revelator:Perkin Elmer UV/VIS Spectrophotometer Lambda 3B 280 nm Column: XK 50Pharmacia (maximum pressure 3 bar) 0 5 cm, height 100 cm Resin:S-Sepharose Pharmacia height 85 cm, volume 1670 ml Eluents: Buffer ASodium acetate 20 mM Urea 4 M pH 5.5, ionic strength 1.5 mS Buffer BSodium acetate 20 mM Urea 4 M pH 5.5 Sodium chloride 1 M ionic strength57.1 mS

All buffers are filtrated by using a Millex pre-filter AP 50 bound inseries with filter 0.45 μm Millipak 20 (Millipore).

-   Temperature: Room temperature-   Conditioning: ≈8000 ml    -   Buffer A: 97% Buffer B: 3% ionic strength mix:    -   4.5 mS    -   Flow: 30 ml/min P=42 PSI    -   Time: 4 hours, 25 minutes-   Loading: 200 g acid casein dissolved in Buffer C (total volume 4000    ml)    -   Flow: 20 ml/min P=50 PSI    -   Time: 3 hours, 20 minutes-   Elution: First Stage (isocratic)≈8000 ml    -   Buffer A:97% Buffer B:3%    -   Flow: 30 ml/min P=42 PSI    -   Time: 4 hours, 25 minutes    -   Second Stage (isocratic) 9000 ml    -   Buffer A:20% Buffer B:80%    -   Flow: 30 ml/min P=42 PSI    -   Time: 5 hours

EXAMPLE 2 Control Test

An amount of the product from example 1 is tested by chromatography toevaluate the absence of beta casein in the isocratic of the secondstage. Such absence is confirmed as demonstrated by the chromatogram ofFIG. 3. By comparison in FIG. 1, the chromatogram relating to theinitial load is illustrated, whereas in FIG. 2 the peak relating to thepresence of beta casein only derived from the elution of the first stageis represented.

Analytic chromatography HPLC Perkin Elmer Biocompatible Binary Pump 250Revelator: Perkin Elmer LC95 280 nm Column: Mono S HR 5/5 PharmaciaLoop: 100 μl Eluents: Buffer A* Sodium acetate 20 mM Urea 6 M pH 5Buffer B* Sodium acetate 20 mM Urea 6 M Sodium chloride 1 M pH 5Temperature Room temperature Flow: 1 ml/min Conditioning: 8′00″ BufferA* Buffer B* 0% 100% Elution: gradient Buffer B* 50% Buffer B* 50% 5′00″(increase of B* 1% min) isocratic 2′00″ Buffer A* 50% Buffer B* 50%isocratic 5′00″ Buffer A*  0% Buffer * 100%

EXAMPLE 3

The Product of Example 1 has been purified from urea by the followingmethod of diafiltration.

-   Ultrafiltration S.G.I.-   Cellulose membrane S-10 10.000 Da Amicon-   Buffer of dialysis: demineralized water    -   sodium acetate 10 mM ph?7    -   ionic strength 0.8 mS    -   total volume 250 l (5 washes)-   Permeate flow: 32+37 l/h-   Temperature: 10° C.-   Product concentration 50 l up to 20 l

The product has been tested to verify the absence of urea as follows:

Urea Test UV method (Boehringer Mannheim) Spectrophotometer: Lambda 3B340 nm (Perkin Elmer) Reagents Blank Sample Solution 1 1.00 ml 1.00 mlSample solution — 0.10 ml Solution 2 0.02 ml 0.02 ml Bidistilled water2.00 ml 1.90 ml Incubate 5′ at 20–25° C.; read the absorbance (A1)Solution 3 0.02 ml 0.02 ml Incubate 20′ at 20–25° C.; read theabsorbance (A2) Solution 1 = Triethanolamin buffer, pH 8.2 oxoglutarate,NADH Solution 2 = Urase Solution 3 = Dehydrogenase glutamate

The lyophilization is carried out on the product free of urea, by usinga Christ model Beta 1–16 equipment.

1. A method for the reducing the occurrence of insulin-dependentdiabetes comprising the step of administering to newborns and infants,who will exhibit in response to feeding of non-human beta casein, animmune response which will cause insulin-dependent diabetes, an infantformula derived from non-human milk, said infant formula beingsubstantially free of non-human beta caseins that exhibit molecularmimicry with protein GLUT 2.